KUZ, a novel family of metalloproteases

ABSTRACT

Members of a novel family of polypeptides, the KUZ family, are metalloproteases involved in neuronal partitioning and neuronal development. The invention provides KUZ poylpeptides, antibodies that bind the KUZ polypeptides, KUZ encoding nucleic acids, methods for identifying cells expressing the KUZ polypeptides, methods of identifying ligands that bind to the subject proteins and methods of blocking KUZ polypeptide/ligand interactions.

This application is a divisional of U.S. Pat. Ser. No. 09/709,126, filedNov. 8, 2000, now U.S. Pat. No. 6,319,704, which is a divisional of U.S.Pat. Ser. No. 09/285,502, filed Apr. 2, 1999, now U.S. Pat. No.6,190,876, which is a divisional of U.S. Pat. Ser. No. 08,937,931, filedAug. 27, 1997, now U.S. Pat. No. 5,935,792 which claims benefit of U.S.Pat. Ser. No. 60/053,476, filed Jul. 23., 1997 and U.S. Pat. Ser. No.60/019,390, filed

FIELD OF THE INVENTION

The field of the invention is a novel family of proteins and genesinvolved in development.

BACKGROUND OF THE INVENTION

Cell-cell interactions play an important role in regulating cell fatedecisions and pattern formation during the development of multicellularorganisms. One of the evolutionarily conserved pathways that plays acentral role in local cell interactions is mediated by the transmembranereceptors encoded by the Notch (N) gene of Drosophila, the lin-12 andglp-1 genes of C. elegans, and their vertebrate homologs (reviewed inArtavanis-Tsakonas, S., et al. (1995) Notch Signaling. Science 268,225-232), collectively hereinafter referred to as NOTCH receptors.Several lines of evidence suggest that the proteolytic processing ofNOTCH receptors is important for their function. For example, inaddition to the full length proteins, antibodies against theintracellular domains of NOTCH receptors have detected C-terminalfragments of 100-120 kd (hereafter referred to as 100 kd fragments); seee.g. Fehon, R. G., et al. (1990). Cell 61, 523-534; Crittenden, S. L.,et al. (1994). Development 120, 2901-2911; Aster. J., et al. (1994) ColdSpring Harbor Symp. Quant. Biol. 59. 125-136; Zagouras, P., etal.(1995). Proc. Natl. Acad. Sci. USA 92, 6414-6418; and Kopan, R., etal. (1996). Proc. Natl. Acad. Sci. USA 93, 1683-1688. However, themechanism(s) of NOTCH activation have been hitherto largely unknown.

During neurogenesis, a single neural precursor is singled out from agroup of equivalent cells through a lateral inhibition process in whichthe emerging neural precursor cell prevents its neighbors from taking onthe same fate (reviewed in Simpson, P. (1990). Development 109,509-519). Genetic studies in Drosophila have implicated a group of“neurogenic genes” including N in lateral inhibition. Loss-of-functionmutations in any of the neurogenic genes result in hypertrophy of neuralcells at the expense of epidermis (reviewed in Campos-Ortega, J. A.(1993) In: The Development of Drosophila melanogaster M. Bate and A.Martinez-Arias, eds. pp. 1091-1129. Cold Spring Harbor Press.). Wedisclose herein a new neurogenic gene family, kuzbanian (kuz) (Rooke,J., Pan, D. J., Xu, T. and Rubin, G. M. (1996). Science 273, 1227-1231).Members of the disclosed KUZ family of proteins are shown to belong tothe recently defined ADAM family of transmembrane proteins, members ofwhich contain both a disintegrin and metalloprotease domain (reviewed inWolfsberg, T. G., et al. (1995). J. Cell Biol. 131, 275-278, see alsoBlobel, C. P., et al. (1992). Nature 356, 248-252, 1992;Yagami-Hiromasa, T., et al. (1995). Nature 377, 652-656; Black, R. A.,et al. (1997). Nature 385, 729-733, 1997; and Moss, M. L., et al.(1997). Nature 385, 733-736).

We further disclose herein various engineered mutant forms of native KUZproteins and their activities. We show that mutant KUZ proteins lackingprotease activity interfere with endogenous KUZ activity and function asdominant negatives (indicating that the protease activity of native KUZis essential to its biological functions) and that dominant negativescan perturb lateral inhibition during neurogenesis and result in theoverproduction of primary neurons. We also show that proteolyticprocessing of NOTCH in embryos to generate the 100 kd species fails tooccur in the kuz mutant embryo and expression of dominant negatives inimaginal discs or tissue culture cells blocks NOTCH processing(indicating that the primary NOTCH translation product isproteolytically cleaved by native KUZ proteins as part of the normalbiosynthesis of a functional NOTCH receptor).

SUMMARY OF THE INVENTION

The invention provides methods and compositions relating to isolated KUZpolypeptides, related nucleic acids, polypeptide domains thereof havingKUZ-specific structure and activity and modulators of KUZ function,particularly Notch protease activity. KUZ polypeptides, nucleic acidsand modulators thereof regulate Notch signal transduction pathways andhence provide important regulators of cell function. The polypeptidesmay be produced recombinantly from transformed host cells from thesubject KUZ polypeptide encoding nucleic acids or purified frommammalian cells. The invention provides isolated KUZ hybridizationprobes and primers capable of specifically hybridizing with thedisclosed KUZ genes, KUZ-specific binding agents such as specificantibodies, and methods of making and using the subject compositions indiagnosis (e.g. genetic hybridization screens for KUZ transcripts),therapy (e.g. KUZ protease inhibitors to modulate Notch signaltransduction) and in the biopharmaceutical industry (e.g. as immunogens,reagents for isolating additional natural kuz alleles, reagents forscreening bio/chemical libraries for ligands and lead and/orpharmacologically active agents, etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A). Sequence alignment of predicted KUZ proteins from Drosophila(DKUZ, SEQ ID NO:2), mouse (MKUZ, SEQ ID NO:8) and Xenopus (XKUZ, SEQ IDNO:10). The full length amino acid sequence of MKUZ was deduced from thenucleotide sequence of two overlapping cDNA clones. Partial amino acidsequence of XKUZ was deduced from the nucleotide sequence of a PCRproduct that includes parts of the disintegrin and Cys-rich domains. Thealignments were produced using Geneworks software (IntelliGenetics).Residues identical among two species are highlighted. Predictedfunctional domains are indicated. Amino acid sequences from whichdegenerate PCR primers were designed are indicated with arrows.Orthologs of kuz are also present in C. elegans (GenBank accession nos.D68061 and M79534), rat (Z48444), bovine (Z21961) and human (Z48579).

FIG. 1(B). Summary of constructs used in this study and theiroverexpression phenotypes. Different domains are indicated by shadings.Asterisks indicate where point mutations were introduced. Constructs 1-9are based on DKUZ, while MKUZDN is based on MKUZ. Abbreviations: ++,strong phenotype; +, weak phenotype; 0, no phenotype.

FIG. 1(C). Schematic diagram of DKUZ, MKUZ and XKUZ. The percentagesgiven refer to sequence identity in the indicated domains between MKUZand either DKUZ or XKUZ.

FIG. 2 shows a schematic of how KUZ protease can process NOTCH on theextracellular domain to generate an N- terminal extracellular fragmentand the C-terminal 100 kd fragment containing the transmembrane and thecytoplasmic domain. These two fragments may remain tethered together tofunction as a competent NOTCH receptor, analogous to the maturation ofthe SEVENLESS receptor (Simon et al., 1989).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides isolated KUZ polypeptides, isolated froma wide variety of sources including Drosophila, human, mouse andXenopus, as well as allelic variants, naturally occurring and alteredsecreted forms, deletion mutants having KUZ-specific sequence and/orbioactivity and mutants comprising conservative amino acidsubstitutions. SEQ ID NOS:1, 3, 5, 7 and 9 depict exemplary naturalcDNAs encoding Drosphila, human transmembrane, human soluble (lacking atransmembrane domain), mouse and Xenopus members, respectively, of thedisclosed KUZ family. SEQ ID NOS: 2, 4, 6, 8 and 10 depict thecorresponding encoded full-length KUZ proteins. Methods used to isolateadditional members of the kuz family are described below and in theExamples.

Preferred translates/deletion mutants comprise at least a 10, preferablyat least a 15, more preferably at least a 20 residue domain of at leastone of SEQ ID NOS:2, 4, 6, 8 and 10. In particular, KUZ derivatives canbe made by altering KUZ sequences by substitutions, additions ordeletions that provide for functionally equivalent molecules. Due to thedegeneracy of nucleotide coding sequences, other DNA sequences whichencode substantially the same amino acid sequence as a kuz gene may beused in the practice of the present invention. These include but are notlimited to nucleotide sequences comprising all or portions of kuz geneswhich are altered by the substitution of different codons that encode afunctionally equivalent amino acid residue within the sequence, thusproducing a silent change. Likewise, the KUZ derivatives of theinvention include, but are not limited to, those containing, as aprimary amino acid sequence, all or part of the amino acid sequence of aKUZ protein including altered sequences in which functionally equivalentamino acid residues are substituted for residues within the sequenceresulting in a silent change. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity which acts as a functional equivalent, resulting in asilent alteration. Conservative substitutes for an amino acid within thesequence may be selected from other members of the class to which theamino acid belongs. For example, the nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine proline, phenylalanine,tryptophan and methionine. The polar neutral amino acids includeglycine, serine, threonine, cysteine, tyrosine, asparagine andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid.

In a specific embodiment of the invention, proteins consisting of orcomprising a fragment of a KUZ protein consisting of at least 10(continuous) amino acids of the KUZ protein is provided. In otherembodiments, the fragment consists of at least 15 or 20 or 50 aminoacids of the KUZ protein. In specific embodiments, such fragments arenot larger than 35, 100 or 200 amino acids. Derivatives or analogs ofKUZ include but are not limited to those peptides which aresubstantially homologous to a KUZ protein or fragments thereof. (e.g.,at least 30%, 50%, 70%, or 90% identity over an amino acid sequence ofidentical size—e.g., comprising a domain) or whose encoding nucleic acidis capable of hybridizing to a coding KUZ sequence.

Ordinarily, the allelic variants, the conservative substitution variantsand the members of the kuz family of proteins, will have an amino acidsequence having at least 75% amino acid sequence identity with one ormore of the disclosed human full length, human secreted form, mouse andDrosophila kuz protein sequences, more preferably at least 80%, evenmore preferably at least 90%, and most preferably at least 95%. Identityor homology with respect to such sequences is defined herein aspercentage of amino acid residues in the candidate sequence that areidentical with the known peptides, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. N-terminal, C-terminal or internal extensions,deletions, or insertions into the peptide sequence shall not beconstrued as affecting homology.

The subject domains provide KUZ domain specific activity or function,such as KUZ-specific protease or protease inhibitory activity,disintegrin or disintegrin inhibitory activity, ligand/antibody bindingor binding inhibitory, immunogenicity, etc.; see, e.g. domainsidentified in FIG. 1A-C. Preferred domains cleave a NOTCH protein.KUZ-specific activity or function may be determined by convenient invitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cellculture assays, in animals (e.g. gene therapy, transgenics, etc.), etc.Binding assays encompass any assay where the molecular interaction of anKUZ polypeptide with a binding target is evaluated. The binding targetmay be a natural intracellular binding target such as an KUZ substrate,a KUZ regulating protein or other regulator that directly modulates KUZactivity or its localization; or non-natural binding target such aspecific immune protein such as an antibody, or an KUZ specific agentsuch as those identified in screening assays such as described below.KUZ-binding specificity may assayed by protease activity or bindingequilibrium constants (usually at least about 10⁷M⁻¹, preferably atleast about 10⁸M⁻¹, more preferably at least about 10M⁻¹, by the abilityof the subject polypeptide to function as negative mutants inKUZ-expressing cells, to elicit KUZ specific antibody in a heterologoushost (e.g a rodent or rabbit), etc. The KUZ binding specificity ofpreferred KUZ polypeptides necessarily distinguishes that of the bovineprotein of Howard, L., et al. (1996). Biochem. J. 317, 45-50.

The claimed KUZ polypeptides are isolated or pure: an “isolated”polypeptide is unaccompanied by at least some of the material with whichit is associated in its natural state, preferably constituting at leastabout 0.5%, and more preferably at least about 5% by weight of the totalpolypeptide in a given sample and a pure polypeptide constitutes atleast about 90%, and preferably at least about 99% by weight of thetotal polypeptide in a given sample. The KUZ polypeptides andpolypeptide domains may be synthesized, produced by recombinanttechnology, or purified from mammalian, preferably human cells. A widevariety of molecular and biochemical methods are available forbiochemical synthesis, molecular expression and purification of thesubject compositions, see e.g. Molecular Cloning, A Laboratory Manual(Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols inMolecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc.,Wiley-Interscience, New York) or that are otherwise known in the art.Material and methods for the expression of heterologous recombinantproteins in bacterial cells (e.g. E. coli), yeast (e.g. S. Cerevisiae),animal cells (e.g. CHO, 3T3, BHK, baculovirus-compatible insect cells,etc.). The KUZ polypeptides and/or domains thereof may be provideduncomplexed with other protein, complexed in a wide variety ofnon-covalent associations and binding complexes, complexed covalentlywith other KUZ or non-KUZ peptide sequences (homo or hetero-chimericproteins), etc.

The invention provides binding agents specific to the claimed KUZpolypeptides, including substrates, agonists, antagonists, naturalintracellular binding targets, etc., methods of identifying and makingsuch agents, and their use in diagnosis, therapy and pharmaceuticaldevelopment. For example, specific binding agents are useful in avariety of diagnostic and therapeutic applications, especially wheredisease or disease prognosis is associated with improper utilization ofa pathway involving the subject proteins. Novel KUZ-specific bindingagents include KUZ-specific receptors, such as somatically recombinedpolypeptide receptors like specific antibodies or T-cell antigenreceptors (see, e.g Harlow and Lane (1988) Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory) and other natural intracellularbinding agents identified with assays such as one-, two- andthree-hybrid screens, non-natural intracellular binding agentsidentified in screens of chemical libraries such as described below,etc. Agents of particular interest modulate KUZ function, e.g.KUZ-dependent proteolytic processing. For example, a wide variety ofinhibitors of KUZ Notch protease activity may be used to regulate signaltransduction involving Notch. Metalloprotease and disintegrin inhibitorsand methods for designing such inhibitors are well known in the art,e.g. Matrisian, L. TIG, 6:(1990), Hooper, N. FEBS Let. 354:1-6 (1994),Haas et al., Cur. Op. Cell Bio. 6:656-662 (1994), etc. Exemplaryinhibitors include known classes of metalloprotease inhibitors,KUZ-derived peptide inhibitor, esp. dominant negative deletion mutants,etc. KUZ specificity, and activity are readily quantified in highthroughput protease assays using panels of proteases.

Accordingly, the invention provides methods for modulating signaltransduction involving Notch in a cell comprising the step of modulatingKUZ protease activity, e.g. by contacting the cell with a proteaseinhibitor. The cell may reside in culture or in situ, i.e. within thenatural host. For use in methods applied to cells in situ, thecompositions frequently further comprise a physiologically acceptableexcipient and/or other pharmaceutically active agent to formpharmaceutically acceptable compositions. Hence, the invention providesadministratively convenient formulations of the compositions includingdosage units which may be incorporated into a variety of containers. Thesubject methods of administration generally involve contacting the cellwith or administering to the host an effective amount of the subjectcompounds or pharmaceutically acceptable compositions. The compositionsand compounds of the invention and the pharmaceutically acceptable saltsthereof can be administered to a host in any effective way such as viaoral, parenteral or topical routes. Preferred inhibitors are orallyactive in mammalian hosts.

In one embodiment, the invention provides the subject compounds combinedwith a pharmaceutically acceptable excipient such as sterile saline orother medium, gelatin, an oil, etc. to form pharmaceutically acceptablecompositions. The compositions and/or compounds may be administeredalone or in combination with any convenient carrier, diluent, etc. andsuch administration may be provided in single or multiple dosages.Useful carriers include solid, semi-solid or liquid media includingwater and non-toxic organic solvents. In another embodiment, theinvention provides the subject compounds in the form of a pro-drug,which can be metabolically converted to the subject compound by therecipient host. A wide variety of pro-drug formulations are known in theart. The compositions may be provided in any convenient form includingtablets, capsules, lozenges, troches, hard candies, powders, sprays,creams, suppositories, etc. As such the compositions, inpharmaceutically acceptable dosage units or in bulk, may be incorporatedinto a wide variety of containers. For example, dosage units may beincluded in a variety of containers including capsules, pills, etc.

The compositions may be advantageously combined and/or used incombination with other therapeutic or prophylactic agents, differentfrom the subject compounds. In many instances, administration inconjunction with the subject compositions enhances the efficacy of suchagents, see e.g. Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9^(th) Ed., 1996, McGraw-Hill. For diagnostic uses, theinhibitors or other KUZ binding agents are frequently labeled, such aswith fluorescent, radioactive, chemiluminescent, or other easilydetectable molecules, either conjugated directly to the binding agent orconjugated to a probe specific for the binding agent.

According to the invention, a KUZ protein, its fragments or otherderivatives, or analogs thereof, may be used as an immunogen to generateantibodies which recognize such an immunogen. Such antibodies includebut are not limited to polyclonal, monoclonal, chimeric, single chain,Fab fragments, and an Fab expression library. In a specific embodiment,antibodies to human KUZ are produced. In another embodiment, antibodiesto the extracellular domain of KUZ are produced. In another embodiment,antibodies to the intracellular domain of KUZ are produced.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to a KUZ protein or derivative or analog. In aparticular embodiment, rabbit polyclonal antibodies to an epitope of theKUZ protein encoded by a sequence selected from SEQ ID NOS: 1, 3, 5, 7or 9 or a subsequence thereof, can be obtained. For the production ofantibody, various host animals can be immunized by injection with thenative KUZ protein, or a synthetic version, or derivative (e.g.,fragment) thereof, including but not limited to rabbits, mice, rats,etc. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, and including but not limitedto Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a KUZ proteinsequence or analog thereof, any technique which provides for theproduction of antibody molecules by continuous cell lines in culture maybe used. For example, the hybridoma technique originally developed byKohler and Milstein (1975, Nature 256: 495-497), as well as the triomatechnique. The human B-cell hybridoma technique (Kozbor et al., 1983,Immunology Today 4:72), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., 1985, in Monoclonal antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additionalembodiment of the invention, monoclonal antibodies can be produced ingerm-free animals utilizing recent technology (PCT/US90/02545).According to the invention, human antibodies may be used and can beobtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad.Sci. U.S.A. 80: 2026-2030) or by transforming human B cells with EBVvirus in vitro (Cole et al., 1985, in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, pp. 77-96). In fact, according to the invention,techniques developed for the production of “chimeric antibodies”(Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing the genes from a mouse antibody moleculespecific for KUZ together with genes from a human antibody molecule ofappropriate biological activity can be used; such antibodies are withinthe scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce KUZ-specific single chain antibodies. An additional embodimentof the invention utilizes the techniques described for the constructionof Fab expression libraries (Huse et al., 1989, Science 246:1275-1281)to allow rapid and easy identification of monoclonal Fab fragments withthe desired specificity for KUZ proteins, derivatives, or analogs.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab′ fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragment, andthe Fab fragments which can be generated by treating the antibodymolecule with papain and a reducing agent. In the production ofantibodies, screening for the desired antibody can be accomplished bytechniques known in the art e.g. ELISA (enzyme-linked immunosorbentassay). For example, to select antibodies which recognize a specificdomain of a KUZ protein, one may assay generated hybridomas for aproduct which binds to a KUZ fragment containing such domain. Forselection of an antibody immunospecific to human KUZ, one can select onthe basis of positive binding to human KUZ and a lack of binding to aKUZ of another species. The foregoing antibodies can be used in methodsknown in the art relating to the localization and activity of theprotein sequences of the invention, e.g., for imaging these proteins,measuring levels thereof in appropriate physiological samples, indiagnostic methods, etc. Antibodies specific to a domain of a KUZprotein are also provided. In a specific embodiment, antibodies whichbind to a Notch-binding fragment of KUZ are provided.

The amino acid sequences of the disclosed KUZ polypeptides are used toback-translate KUZ polypeptide-encoding nucleic acids optimized forselected expression systems (Holler et al. (1993) Gene 136, 323-328;Martin et al. (1995) Gene 154, 150-166) or used to generate degenerateoligonucleotide primers and probes for use in the isolation of naturalKUZ-encoding nucleic acid sequences (“GCG” software, Genetics ComputerGroup, Inc, Madison Wis.). KUZ-encoding nucleic acids used inKUZ-expression vectors and incorporated into recombinant host cells,e.g. for expression and screening, transgenic animals, e.g. forfunctional studies such as the efficacy of candidate drugs for diseaseassociated with KUZ-modulated cell function, etc.

The invention also provides nucleic acid hybridization probes andreplication/amplification primers having a KUZ cDNA specific sequencecomprising SEQ ID NO: 1, 3, 5, 7 or 9, and sufficient to effect specifichybridization thereto (i.e. specifically hybridize with SEQ ID NO: 1, 3,5, 7 or 9, respectively, in the presence of an embryonic cDNA libraryfrom the corresponding species, and preferably in the presence of BMPcDNA as described by Howard and Glynn (1995). Such primers or probes areat least 12, preferably at least 24, more preferably at least 36 andmost preferably at least 96 bases in length. Demonstrating specifichybridization generally requires stringent conditions, i.e. those that(1) employ low ionic strength and high temperature for washing, forexample, 0.015 M NaCl/0.0015 M sodium titrate/0.1% SDS at 50° C., or (2)employ during hybridization a denaturing agent such as formamide, forexample, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM NaCl, 75 mM sodium citrate at 42° C. Another example is useof 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 (g/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1%SDS. KUZ nucleic acids can also be distinguished using alignmentalgorithms, such as BLASTX (Altschul et al. (1990) Basic Local AlignmentSearch Tool, J Mol Biol 215, 403-410).

The subject nucleic acids are of synthetic/non-natural sequences and/orare isolated, i.e. unaccompanied by at least some of the material withwhich it is associated in its natural state, preferably constituting atleast about 0.5%, preferably at least about 5% by weight of totalnucleic acid present in a given fraction, and usually recombinant,meaning they comprise a non-natural sequence or a natural sequencejoined to nucleotide(s) other than that which it is joined to on anatural chromosome. Recombinant nucleic acids comprising the nucleotidesequence of SEQ ID NO: 1, 3, 5, 7 or 9, or the subject fragmentsthereof, contain such sequence or fragment at a terminus, immediatelyflanked by (i.e. contiguous with) a sequence other than that which it isjoined to on a natural chromosome, or flanked by a native flankingregion fewer than 10 kb, preferably fewer than 2 kb, which is at aterminus or is immediately flanked by a sequence other than that whichit is joined to on a natural chromosome. While the nucleic acids areusually RNA or DNA, it is often advantageous to use nucleic acidscomprising other bases or nucleotide analogs to provide modifiedstability, etc.

The subject nucleic acids find a wide variety of applications includinguse as translatable transcripts, knock-in/out vectors, hybridizationprobes, PCR primers, diagnostic nucleic acids, etc.; use in detectingthe presence of KUZ genes and gene transcripts and in detecting oramplifying nucleic acids encoding additional KUZ homologs and structuralanalogs. In diagnosis, KUZ hybridization probes find use in identifyingwild-type and mutant KUZ alleles in clinical and laboratory samples.Mutant alleles are used to generate allele-specific oligonucleotide(ASO) probes for high-throughput clinical diagnoses. In therapy,therapeutic KUZ nucleic acids are used to modulate cellular expressionor intracellular concentration or availability of active KUZ.

The invention provides efficient methods of identifying agents,compounds or lead compounds for agents active at the level of a KUZmodulatable cellular function. Generally, these screening methodsinvolve assaying for compounds which modulate KUZ interaction with anatural KUZ binding target such as a Notch protein, etc. A wide varietyof assays for binding agents are provided including labeled in vitroprotein-protein binding assays including protease assays, immunoassays,cell based assays, etc. The methods are amenable to automated,cost-effective high throughput screening of chemical libraries for leadcompounds. Identified reagents find use in the pharmaceutical industriesfor animal and human trials; for example. The reagents may bederivatized and rescreened in vitro and in vivo assays to optimizeactivity and minimize toxicity for pharmaceutical development.

Exemplary in vitro binding assays employ a mixture of componentsincluding an KUZ polypeptide, which may be part of a fusion product withanother peptide or polypeptide, e.g. a tag for detection or anchoring,etc. The assay mixtures comprise a natural intracellular KUZ bindingtarget. In a particular embodiment, the binding target is a Notchprotein-derived substrate of KUZ protease activity. Such substratescomprise a specifically KUZ-cleavable peptide bond and at least 5,preferably at least 10, and more preferably at least 20 naturallyoccurring immediately flanking residues on each side. While nativefull-length binding targets may be used, it is frequently preferred touse portions (e.g. peptides) thereof so long as the portion providesbinding affinity and avidity to the subject KUZ polypeptide convenientlymeasurable in the assay. The assay mixture also comprises a candidatepharmacological agent. Candidate agents encompass numerous chemicalclasses, though typically they are organic compounds; preferably smallorganic compounds and are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. A variety ofother reagents may also be included in the mixture. These includereagents like ATP or ATP analogs (for protease assays), salts, buffers,neutral proteins, e.g. albumin, detergents, non-specific proteaseinhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.

The resultant mixture is incubated under conditions whereby, but for thepresence of the candidate pharma ologic agent, the KUZ polypeptidespecifically binds the cellular binding target portion or analog with areference binding affinity. The mixture components can be added in anyorder that provides for the requisite bindings and incubations may beperformed at any temperature which facilitates optimal binding.Incubation periods are likewise selected for optimal binding but alsominimized to facilitate rapid, high-throughput screening.

After incubation, the agent-biased binding between the KUZ polypeptideand one or more binding targets is detected by any convenient way. ForKUZ protease assays, ‘binding’ is generally detected by the generationof a KUZ substrate cleavage product. In this embodiment, proteaseactivity may quantified by the apparent transfer a label from thesubstrate to the nascent smaller cleavage product, where the label mayprovide for direct detection as radioactivity, luminescence, optical orelectron density, etc. or indirect detection such as an epitope tag,etc. A variety of methods may be used to detect the label depending onthe nature of the label and other assay components, e.g. through opticalor electron density, radiative emissions, nonradiative energy transfers,etc. or indirectly detected with antibody conjugates, etc.

A difference in the binding affinity of the KUZ polypeptide to thetarget in the absence of the agent as compared with the binding affinityin the presence of the agent indicates that the agent modulates thebinding of the KUZ polypeptide to the KUZ binding target .Analogously,in cell-based assays described, a difference in KUZ-dependent modulationof signal transduction in the presence and absence of an agent indicatesthe agent modulates KUZ function. A difference, as used herein, isstatistically significant and preferably represents at least a 50%, morepreferably at least a 90% difference.

Altered Drosophila hosts in which the kuz gene is over-expressed,under-expressed, mis-expressed or expressed as a variant are used toidentify compounds that are antagonist or agonists of the KUZpolypeptide as well as to identify genes that encode products thatinteract with the KUZ polypeptide using art known methods (Xu et al.,Genes and Devel., p464-475 (1990), Simon et al., Cell, 67:701-716 (1991)and Fortini et al., Cell, 79:273-282 (1994)).

Agents that modulate the interactions of the KUZ polypeptide with itsligands/natural binding targets can be used to modulate biologicalprocesses associated KUZ function, e.g. by contacting a cell comprisinga KUZ polypeptide (e.g. administering to a subject comprising such acell) with such an agent. Biological processes mediated by KUZpolypeptides include a wide variety of cellular events which aremediated when a KUZ polypeptide binds a ligand e.g. celldifferentiation, cell development and neuronal partitioning. The agentsare also used to modulate processes effected by KUZ substrates; forexample, Notch, an art known peptide involved in neurogenesis isover-expressed in some forms of leukemia (Ellison et al., Cell,66:649-661 (1991)).

The present invention further provides methods for identifying cellsinvolved in KUZ polypeptide-mediated activity, e.g. by determiningwhether the KUZ polypeptide, or a kuz ligand, is expressed in a cell.Such methods are useful in identifying cells and events involved inneurogenesis. In one example, an extract of cells is prepared andassayed by of a variety of immunological and nucleic acid techniques todetermine whether the KUZ polypeptide is expressed. The presence of theKUZ polypeptide provides a measurement of the participation or degree ofneurogenesis of a cell.

The invention provides a wide variety of methods and compositions forevaluating modulators of the KUZ signaling pathways. For example, theinvention provides transgenic non-human animals such as flies (e.g.Drosophila), worms (e.g. C. elegans), mice, etc. having at least onestructurally and functionally disrupted KUZ allele. In particularembodiments, the animals comprise a transgene within a KUZ allele locus,encoding a selectable marker and displacing at least one exon of the KUZallele. More particularly, the transgene may comprise 3′ and 5′ regionswith sufficient complementarity to the natural KUZ allele at the locusto effect homologous recombination of the transgene with the KUZ allele.Such animals provide useful models for determining the effect ofcandidate drugs on a host deficient in KUZ function.

As describe above, the invention provides a wide variety of methods formaking and using the subject compositions. As additional examples, theinvention provides methods for determining the effect of a candidatedrug on a host deficient in KUZ function, such as: contacting atransgenic animal having at least one disrupted KUZ allele with acandidate drug; and, detecting the presence or absence of aphysiological change in the animal in response to the contacting step.The invention also provides methods of evaluating the side effects of aKUZ function inhibitor, such as: contacting a transgenic animal havingat least one disrupted KUZ allele with a candidate drug; detecting thepresence or absence of a physiological change in the animal in responseto the contacting step, wherein the presence of a physiological changeindicates the inhibitor has side effects beyond KUZ function inhibition.

Without further description, one of ordinary skill in the art can, usingthe preceding description and the following illustrative examples, makeand utilize the compounds of the present invention and practice theclaimed methods. The following working examples therefore, specificallypoint out preferred embodiments of the present invention, and are not tobe construed as limiting in any way the remainder of the disclosure.Other generic configurations will be apparent to one skilled in the art.All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

EXAMPLES Example 1 Identification of a Drosophila KUZ Polyleptide/gene

Genes involved in lateral inhibition were screened using FLP/FRTchromosomes to produce mutant clones in mosaic animals (T. Xu and G. M.Rubin, Development 117:1223 (1993); T. Xu and S. Harrison Methods inCell Biology 44:655 (1994)) and to isolate several alleles of a genefamily designated herein as kuzbanian(kuz). The kuz locus is defined bya single complementation group which maps to chromosomal location34C4,5, and corresponds to the 1(2)34 Da group (A. C. Spradling et al.,PNAS 92:10824 (1995). Most of the kuz phenotypic analysis was performedusing the null allele kuze29-4. Kuze29-4 is an excision allele deletingapproximately 2.4 kb at the 5′ end of the kuz gene, including DNA in thepromoter region and the first and second exons. Four P[lacZ; w+]insertions 1(2)k11804, 1(2)k01403, 1(2)k07601 and 1(2)k14701 arehypomorphic kuz alleles. The insert either in the first kuz exon or inthe first intron. Precise excision of these P insertions reverts theassociated kuz phenotype. Kuz1 is the original kuz allele caused by aninsertion of 4.3 kb of DNA in or near the first exon. Seventeenadditional X-ray induced kuz alleles were isolated in the FLP/FRT mosaicscreen.

A 10 kb fragment of DNA from the region deleted in allele kuze29-4 wasused to screen a Drosophila total imaginal disc cDNA library. A group oftwo overlapping 1.2 kb cDNAs mapping to this region was recovered; afull-length kuz cDNA, NB1, was isolated from an embryonic cDNA libraryusing the small cDNA clones as probes (Kuz cDNA Genbank accessionnumber: U6059 1).

Scanning electron microscopy (SEM) and embryo staining and adult eyesections were carried out following standard procedures (A. Tomlinsonand D. F. Ready, Dev. Biol. 123:264 (1987); T. Xu and S.Artavanis-Tsakonas, Genetics 126, 665 (1990)). A scanning electronmicrograph (SEM) showing the multiple bristle phenotype in an adultmosaic fly with homozygous kuz clones revealed that aeveral macro- andmicrochaete positions have supernumerary bristles whereas others aremissing in the same area. SEMs showing kuz clones in the eye revealedthe regular array of ommatidia is severely disrupted, that toward thecenter of the clone the density of photoreceptors is abnormally low andnone are successfully organized into ommatidia, and that chimericommatidia at the clone border contain a mixture of pigmented wild-typephotoreceptor cells and mutant, unpigmented photoreceptors. Confocalimages of embryos stained with the neuronal-specific anti-Elav antibodydemonstrate a requirement for maternal and zygotic kuz products. A kuzmaternal null embryo (generated using the ovoD mutation as described inT. B. Chou and N. Perrimon, Genetics 131:643 (1992)) with one zygoticcopy of kuz revealed that a greater proportion of the embryo developedas neural tissue than in wild-type and a surface view of a kuz nullembryo with no maternal or zygotic kuz product showed that most cellsadopted a neural fate. A lower focal plane of this same embryo showedthat all cells around the periphery of the embryo are neural cells. Acuticular preparation of a kuz maternal null embryo with one zygoticcopy of kuz showed a small patch of cuticle develops on the dorsal sideof the embryo; presumably the remaining cells which failed to producecuticle adopted a neural fate, consistent with the previously phenotype.A cuticular preparation of a kuz null embryo showed only a tiny dot ofcuticle developed. Most of these embryos show no cuticle at all.

Animals with kuz mutant clones exhibit clusters of sensory bristles atpositions in which single sensory bristles are normally observed.Separate sockets are often seen with individual bristles, andstimulation of mutant bristles in a reflex test elicits a leg cleaningresponse, indicating that mutant clusters contain multiple sensorybristles and not just multiple shafts (P. Vandervorst and A. Ghysen,Nature 286:65 (1980)). This multiple bristle phenotype is observed inclones mutant for several neurogenic genes such as Notch (N) and shaggy(sgg, also known as zeste-white 3), and is indicative of a failure oflateral inhibition during the development of the peripheral nervoussystem (S. Artavanis-Tsakonas, et al., Trends in Genetics 7:403 (1991);J. S. Campos-Ortega (1993); Jan, Y. N. and Jan, L. Y., id, pp.1207-1244; Romani, S. et al., Genes Dev. 3:997 (1989);Artavanis-Tsakonas, S. et al., Science 268:225 (1995); Heitzler, P. andSimpson, P. (1991). Cell 64, 1083-1092).

Unlike the N phenotype, kuz clones do not produce ectopic bristles,indicating kuz is not required for correct spacing between proneuralclusters. Mutant clones in the adult eye severely disrupted the regulararray of ommatidia. Thin sections through such a mosaic eye reveal thatmutant photoreceptors are not organized correctly into ommatidia.

To determine whether the KUZ polypeptide is required for the developmentof the central nervous system (CNS), embryos lacking any maternallyderived KUZ polypeptide and containing one or no zygotic copies of thekuz gene were produced. The embryos were examined by staining withneuronal-specific antibodies to the Elav protein (Bier, E. et al.,Science 240:913 (1988); Robinow, S. et al., J. Neurobiol. 22,443(1991)). Maternal null embryos with one copy of zygotic kuz geneshowed hyperplasia and disorganization of the CNS on the ventral side ofthe embryos, which is a phenotype similar to the neurogenic phenotype ofN mutant embryos (Lehmann, R. et al., Roux's Arch Dev. Biol. 192:62(1983)). However, embryos lacking all maternal and zygotic KUZpolypeptide have a much more severe neurogenic phenotype. Hypertrophy ofthe nervous system is not restricted to the ventral region, but theembryos stained throughout with anti-Elav, demonstrating that many morecells in the embryo had developed as neural cells. Such a severeneuralizing phenotype is similar to that of sgg null embryos (Bourouis,M. et al., Nature 341:442 (1989)). Cuticular preparation of embryoscorrelated well with the antibody results: Maternal-null embryos withone copy of the kuz gene produced a small patch of cuticle on the dorsalside, consistent with the observation that many of the ventral cells hadadopted a neural fate at the expense of epidermis. Embryos with no KUZpolypeptide produced little or no cuticle, as would be expected if mostcells had become neural, leaving few epidermal cells to secrete cuticle.

Further analyses on the development of adult sensory bristles wereperformed to determine a specific role for the KUZ polypeptide inlateral inhibition. The yellow (y) and crinkle (ck) marker mutationswere used to mark kuz- clones in the adult cuticle. This allows one todetermine the genotype of individual cells and thus to examine theautonomy of the kuz mutant phenotype. Such analysis can distinguishbetween sending and receiving roles for a gene involved in the lateralinhibition process (Heitzler, P. et al., Cell 64:1083 (1991)).

A role for the KUZ polypeptide in lateral inhibition is suggested by theobservation that all sensory bristles in a mutant cluster are kuz-; nowild-type bristles are ever present in a cluster. SEM of kuz- clones(each kuz- cell is also ck- and y-) revealed that the ck- mutationresults in extra trichomes in the epidermnal cell and in blunted shaftsof sensory bristles; these morphological changes allow the borderbetween mutant and wild-type cells to be precisely determined. A markedabsence of all micro- and macrochaetes is observed in the interior ofthe clone, as no shafts, sockets, or neurons (naked cells) are seen.Kuz- mutant cells at normal bristle positions do form bristles at cloneborders where they are in contact with wild-type cells. A high-magnification view of one of the multiple macrochaete clusters at aclone border revealed that every bristle in this and other clusters isalways ck- and y-, demonstrating that all bristles in a cluster arekuz-. No wild-type bristles are observed in multiple bristle clusters.Marked kuz- clones were generated in y- w-hsFLP1;kuse29-4ck-P[FRT]40A/P[y+]P[w+]P[FRT]40A first instar larvae followingprotocols described in T. Xu and G. M. Rubin, Development 117:1223(1993) and T. Xu and S. Harrison Methods in Cell Biology 44:655 (1994).

Mosaic analysis for kuz- clones in the adult cuticle indicates twodistinct functions for the kuz protein. First, the failure of lateralinhibition, evidenced by the formation of extra bristles, only occurs inkuz- mutant cells. This cell-autonomous mutant phenotype indicates thatduring normal development, the kuz protein is required in cells toreceive an inhibitory signal. Kuz- cells at normal bristle-formingpositions become bristles only when they are in contact with wild-typecells, indicating that in wild-type animals, the KUZ polypeptide may actas a positive signal or is involved in sending a positive signal for thedevelopment of the bristle. Thus, there is a cell-autonomous requirementfor kuz in order for cells to be inhibited from adopting a neuralprecursor fate. We conclude that the KUZ polypeptide is required incells to receive an inhibitory signal from the emerging neural cell.Cells in the proneural cluster with wild-type KUZ polypeptide functionreceive the inhibitory signal and are forced to become epidermal,whereas kuz- cells cannot be inhibited and develop as neural precursorcells. A second distinct role for the KUZ polypeptide was revealed bythe same mosaic analyses. All mutant bristle clusters were produced atclone borders, where mutant cells contact wild-type cells. No bristleswere ever produced in clone interiors, either singly or in clusters.Large kuz-clones therefore cause bare patches devoid of bristlescontaining only hair-secreting epidermal cells. This phenotype indicatesthere is a non cell-autonomous requirement for the KUZ polypeptide inbristle development. Hence, Kuz participates in both neural-promotingand -inhibiting processes during formation of the adult epidermis.

To reveal the molecular basis of the KUZ polypeptide functions, a kuzgene was cloned and a full-length cDNA was obtained. The kuz cDNAcontained an open reading frame that encodes a 1,239 amino acidmembrane-spanning protein of the metalloprotease-disintegrin familyknown as the ADAM family (members of the ADAM family contain “ADisintegrin And Metalloprotease Domain”. The KUZ metalloprotease domainalso contains a conserved zinc-binding site (Jiang, W. and Bond, J. S.(1992). FEBS Letters 312, 110-114). Like other disintegrins KUZ has acharacteristic spacing of cysteine residues that is required for theirdirect binding to receptors (Niewiarowski, S. et al., Seminars inHematology 31:289 (1994)). These cysteines are conserved in the KUZpolypeptide along with many additional residues that are shared by otherdisintegrin domains. In this family, many proteins with a multi-domainstructure are proteolytically processed to produce multiple peptideswith different functions (Blobel, C. P. et al., J. Cell Biol. 111:69(1990); Neeper, M. P. et al., Nucleic Acids Res. 18:4255 (1990); Au, L.C., et al., Biochem. Biophys. Res. Commun. 181:585 (1991)). Themetalloprotease and disintegrin domains of kuz may be cleaved from thefull-length precursor to produce both soluble and membrane-bound formsof these domains. Such proteolytic products of the KUZ polypeptide maybe used to carry out the different KUZ polypeptide functions.

Example 2 Identification of Two Human and One Mouse KUZPolypelptides/genes

The nucleic acid sequence of the Drosophila kuz gene was used togenerate PCR primers for amplifying kuz encoding nucleic acid moleculesfrom organisms other than Drosophila. A .9 kb cDNA fragment wasamplified from a human fetal brain cDNA library (Clonetech, Stratagene)using PCR primers. This fragment was cloned and was used as a probe toscreen the human fetal brain cDNA library (Clonetech, Stratagene). Aclone containing a 3.5 kb insert was obtained (SEQ ID NO:3). The clonedcontained a full length encoding sequence that encodes a protein of 749amino acids. Three additional clones were obtained that showed variantrestriction digestion patterns. Sequence analysis of these clonesidentified a second form of the human KUZ polypeptide. This second formof the KUZ polypeptide encodes a protein of 330 amino acids in length(SEQ ID NO:6). A fragment of the human kuz encoding sequence was used toprobe a mouse fetal brain cDNA library. One of four isolated clones wassequenced and contained a 4 kb insert representing a murine KUZ cDNA(SEQ ID NO:7).

Northern blots run using RNA isolated from various mouse and humantissues revealed expression in fetal and adult tissues. Hybridization ofthe blots with probes specific to each of the human forms confirmed thateach of the transcripts was unique to one of the two forms, indicatingthat the two identified mRNA transcripts represent each of the two humanforms respectively. The variable pattern of expression seen on the adultand fetal Northern blots indicates a developmental role of the KUZpolypeptides: the short form being predominant in adult tissues whilethe full length form is predominant in fetal tissues and adult brain.All regions of the adult brain expressed both forms.

Example 3 KUZBANIAN Controls Proteolytic Processing of NOTCH andMediates Lateral Inhibition During Drosophila and VertebrateNeurozenesis

To investigate how the different domains of KUZ contribute to itsbiological functions, full length and various N- and C- terminaltruncations of KUZ were generated (e.g. constructs 1-4 and 7, FIG. 1B)and expressed under the pGMR vector (Hay, B. A., Wolff, T. and Rubin, G.M. (1994). Development 120, 2121-2129) in the developing retina ofDrosophila. One of these exemplary truncations (7), which is missing theprotease domain, resulted in a dominant rough eye phenotype. Weexpressed KUZ truncations using the pDMR vector which contains thedecapentaplegic (dpp) disc specific enhancer element (see experimentalprocedures) that drives gene expression in several tissues includingparts of the notum and the wing blade, two tissues that are known to beaffected in kuz mutant clones. Expression of construct 7 under pDMRresulted in supernumerary bristles on the notums and notches on the wingblades. These phenotypes resemble those seen in somatic cloneshomozygous for kuz loss-of-function mutations, indicating that thisconstruct functions in a dominant negative manner by interfering withendogenous kuz activity. We also observed that the mutant phenotypesresulting from this construct are dominantly enhanced by removing onecopy of the endogenous kuz gene; that is, the phenotypes of kuz/+individuals carrying this construct are more severe than those of +/+individuals. Conversely, additional wildtype KUZ protein from atransgene expressing full length KUZ suppresses these phenotypes. Werefer to the particular dominant negative of construct 7 hereafter asKUZDN (KUZ dominant nnegative).

To directly address the functional relevance of the protease domain, weintroduced into full length KUZ a point mutation (E606 to A) in theputative zinc binding site (FIG. 1A) of the protease domain. Thisglutamate is thought to be a catalytic residue and is absolutelyconserved among all known metalloproteases (Jiang and Bond, 1992). Thus,this point mutation should abolish protease activity while havingminimal impact on the other activities of KUZ. Indeed, overexpression ofKUZ^(E606A) (construct 8 in FIG. 1B) gave similar, although somewhatweaker, dominant phenotypes to those seen with KUZDN.

The notums of Drosophila adults carry two types of sensory bristles,macrochaetes and microchaetes. The sensory organ precursor cells (SOPs)that generate the macrochaetes are selected from pools of equivalentcells by lateral inhibition mostly during the third instar larval stage,while the SOPs for the microchaetes are singled out during the earlypupae stage (Huang, F., et al. (1991). Development 111, 1087-1095;Hartenstein, V. and Posakony, J. W. (1989). Development 107, 389-405). Nis required for this process and removal of N function at larval andpupal stages differentially affects these two types of bristles(Hartenstein, V. and Posakony, J. W. (1990). Dev. Biol. 142, 13-30). IfKUZ is required for lateral inhibition, we would expect to generatesimilar phenotypes by expressing KUZDN at these times. We generatedflies containing KUZDN under the control of the hsp70 promoter, andapplied one hour heat pulses at various times during larval and pupaldevelopment. We observed that while heat pulses applied during thirdinstar larval stage resulted in supernumerary macrochaetes only, heatpulses applied during early pupal stages (0-13 hrs after pupariumformation (APF)) resulted in supernumerary microchaetes only, similar tothe phenotypes resulted from removing N function at these times using atemperature sensitive N allele (Hartenstein and Posakony, 1990). Thesetime points match the periods when SOPs for each bristle type areselected from pools of equivalent cells (Huang et al., 1991; Hartensteinand Posakony, 1989), indicating that KUZDN interferes with lateralinhibition during the selection of SOPs.

kuz mutant clones affect other tissues such as the eye. We perturbed kuzfunctions by expressing KUZDN under the control of the rough enhancer,which drives gene expression in all cells within the morphogeneticfurrow as well as transiently in R2, R5, R3 and R4 posterior to thefurrow (Heberlein, U., et al. (1994). Mech. Dev. 48, 35-49). Fliescarrying the rough/KUZDN transgene had supernumerary photoreceptor cellsin each ommatidium. Neuronal differentiation in these transgenic flieswas followed by staining for ELAV, a neuronal marker, in eye imaginaldiscs. Consistent with the adult eye phenotype, we observed therecruitment of extra neurons into each ommatidial cluster in thedeveloping retina. These experiments indicate that kuz function isrequired to limit the number of photoreceptor neurons recruited intoeach ommatidium.

Besides its functions in determining neural fate, kuz is also requiredfor axonal extension at later stages of neural development (Fambrough,D., et al. (1996). Proc. Natl. Acad. Sci. USA 93, 13233-13238). Weexpressed KUZDN under the control of the ELAV promoter using theGAL4-UAS system (Brand, A. H., and Perrimon, N. (1993). Development 118,401-415). The ELAV promoter drives gene expression in maturing andmature neurons, but not neuroblasts, thus allowing one to bypass therequirement for kuz in neural fate determination. We observed thatembryos expressing KUZDN in developing neurons show major defects inaxonal pathways, such as disruption of longitudinal axonal tracts. Ingeneral, this phenotype is similar to the that observed in zygotic kuzmutant embryos (Fambrough et al., 1996), indicating that KUZ provides aproteolytic activity synthesized by axons and required by them to extendgrowth cones through the extracellular matrix.

Database searches revealed sequences representing putative kuz orthologsin C. elegans, rat, bovine and human. The bovine homolog was initiallyisolated as a proteolytic activity on myelin basic protein in vitro(Howard et al., 1996). We isolated and sequenced cDNAs representing afull-length mouse kuz homolog. This mouse protein (MKUZ) is 45%identical in primary sequence with Drosophila KUZ (DKUZ, FIG. 1), and95% identical with the bovine protein. Sequence similarity between MKUZand DKUZ extends over the whole coding region, except that MKUZ, likeother vertebrate KUZ homologs, has a much shorter intracellular domain.The intracellular domain of MKUZ contains a stretch of 9 amino acidresidues (934-942) that are absolutely conserved with DKUZ. To determinethe functional importance of this sequence similarity, we introducedinto KUZDN mutations in several these mutations dramatically reducedKUZDN activity.

Based on the structure of KUZDN described above, we engineered adominant negative form of MKUZ (MKUZDN, FIG. 1B) missing the proteasedomain. When overexpressed in Drosophila using the pDMR vector, MKUZDNresulted in dominant phenotypes resembling those created by itsDrosophila counterpart. To test directly the involvement of MKUZ invertebrate neurogenesis, we injected in vitro transcribed mRNA encodingMKUZDN into Xenopus embryos. Primary neurons in Xenopus are generated inprecise and simple patterns and can be identified by their expression ofa neural specific β-tubulin gene (N-tubulin). This assay has been usedpreviously to demonstrate a conserved role for certain neurogenic genesin singling out primary neurons in Xenopus by lateral inhibition(Chitnis, A., et al. (1995). Nature 375, 761-766). If a kuz-likeactivity is required for the lateral inhibition process in Xenopus, wewould expect interference with this endogenous kuz activity to result inthe overproduction of primary neurons. Indeed, injection of mRNAencoding MKUZDN resulted in extra N-tubulin positive cells. Consistentwith the notion that kuz acts to limit the number of cells thatdifferentiate as neurons from a group of competent cells, these extraN-tubulin positive cells were confined to domains of primaryneurogenesis, and-were not observed at ectopic positions.

To provide further evidence for an endogenous kuz activity duringprimary neurogenesis in Xenopus, we examined the expression pattern of aXenopus kuz homolog (Xkuz). A cDNA fragment representing a portion ofXkuz (FIG. 1) was isolated (see experimental procedures) and used togenerate RNA probes for in situ hybridization under high stringency.Xkuz is expressed uniformly in gastrulating and neural plate stageembryos, including the domains of primary neurogenesis. In olderembryos, Xkuz continues to be widely expressed, with an elevated levelin neural tissues. Thus, Xkuz is expressed at the appropriate time andplace for a potential role in primary neurogenesis in Xenopus.

We sought to determine the order of action of N and kuz by examining thephenotype produced by combining a gain-of-function N mutant and aloss-of-function kuz mutant. Expression of an activated form of NOTCH(reviewed in Artavanis-Tsakonas et al., 1995) under the heat shockpromoter (hs-N^(act)) at early pupal stages (7-9 hours APF) leads to theloss of microchaetes on the notum; the opposite phenotype, extramicrochaetes, is seen in loss-of-function kuz mutant clones. We focusedon microchaetes since the SOPs for these bristles are generated moresynchronously than those of the macrochaetes (Huang et al., 1991;Hartenstein and Posakony, 1989) and thus a single pulse of heatshock at7-9 hrs APF results in the reproducible loss of microchaetes on thenotum in hs-N^(act) flies. If kuz acts genetically downstream of N, thenthe combination of N^(act) and kuz should display the kuz phenotype ofextra microchaetes. Conversely, if kuz acts genetically upstream of N,then the combination of N^(act) and kuz should display the N^(act)phenotype of missing microchaetes. We observed that the combination ofN^(act) and kuz displayed the N^(act) phenotype, indicateing that kuzacts genetically upstream of N. This result indicates KUZ acts upstreamof, or parallel with NOTCH in the same biochemical pathway.

We observed dosage sensitive genetic interactions between kuz and N,indicating that the levels of activity of kuz and N are tightlybalanced. We took advantage of a weak dpp-KUZDN transgene that resultedin an average of 3 posterior scutellar bristles instead of the 2 seen inwildtype. While heterozvgous N mutants have normal number of posteriorscutellar bristles, this genetic background dramatically enhanced thephenotype resulting from the weak dpp-KUZDN transgene such that anaverage of 5.2 bristles (n=50) were observed. Furthermore, in flies thatcar an additional copy of N gene, the extra bristle phenotype resultingfrom this KUZDN transgene is completely suppressed such that 2 bristleswere observed. This intricate balance between their activities indicatesthat kuz and N are closely linked in a common biological process.

We examined if perturbation of KUZ function in Drosophila Schneider 2(S2) cell cultures would affect NOTCH processing. S2 cells do notexpress any endogenous NOTCH protein (Fehon et al., 1990), but doexpress high levels of kuz mRNA. Upon transfection of a full-length Nconstruct, the monoclonal antibody C17.9C6, which was raised against theintracellular domain of NOTCH, can detect full length NOTCH (about 300kd) and C-terminal fragments of about 100 kd (Fehon et al., 1990). Wereasoned that if kuz is involved in generating this 100 kd species in S2cells, then expression of KUZDN might interfere with this proteolyticevent. Indeed, expression of KUZDN nearly abolished the 100 kd speciesin S2 cells, while the 300 kd species was not greatly affected,indicating that kuz is required for the NOTCH processing. Consistentwith our results in transgenic flies that overexpression of full lengthKUZ did not perturb neurogenesis, transfection of a full length KUZconstruct did not affect NOTCH processing in S2 cells.

Next, we performed similar experiments in developing imaginal discs. Asdescribed earlier, in transgenic flies containing KUZDN under thecontrol of the heatshock promoter, one hour heatshock at the thirdinstar larval stage resulted in extra bristles on the notum. The sameheatshock regime also resulted in notches on the wing blade and extraphotoreceptors in the eye. We followed the status of NOTCH processing inthe wing and eye imaginal discs after the induction of KUZDN in theseanimals. As in transfected S2 cells, mAb C 17.9C6 normally detects a 300kd and a 100 kd NOTCH species in protein extracts of the third instarimaginal discs. After the induction of KUZDN by one hour heatshock, the100 kd species gradually disappears; by 4 hours after induction, the 100kd species is almost undetectable, while the 300 kd species hasaccumulated to a higher level. By 15 hrs after the heatshock, the 100 kdspecies is restored to wildtype levels presumably reflecting the decayof the KUZDN protein synthesized in response to the heatshock. Thecorrelation between the reduction of the 100 kd species upon KUZDNexpression and the resulting neurogenic phenotypes in imaginal tissuesindicates the functional significance of the 100 kd NOTCH form detectedin vivo.

Finally, we examined NOTCH processing in kuz null mutant embryos. Sincekuz is known to have a maternal contribution (supra), we generatedgermline clones to obtain embryos lacking all KUZ function. We foundthat while mAb C17.9C6 detects a 300 kd and a 100 kd species in wildtypeembryos, only the 300 kd species is detected in kuz null embryos. Thisobservation indicates that the phenotypes we generated by expression ofKUZDN are not due to interference with genes other than kuz, such asother members of the ADAM family, and that kuz is required for theproteolytic processing of NOTCH (FIG. 2).

Our studies provide a general scheme for engineering dominant negativeforms of ADAM proteins applicable to other ADAM genes. While all ADAMspossess a disintegrin-like and a metalloprotease-like domain, some ADAMslack a consensus active site in the metalloprotease domain. These“protease dead” ADAMs resemble dominant negative forms of KUZ describedherein and can function as endogenous inhibitors.

Experimental Procedures: Plasmid Constructs: We initially used the pGMRvector (Hay et al., 1994) to express full length KUZ and several N- andC-terminal deletion constructs in the eye. These constructs include 1,2, 3, 4 and 7. Upon identification of 7 as a dominant negative form(KUZDN), we then used another expression vector pDMR to expressconstructs 1, 4, 5, 6, 7, 8 and 9. The pDMR vector utilizes the dpp discspecific enhancer to drive gene expression in multiple tissues includingthe wing and the notum. pDMR was constructed by the following steps.First, the heat shock responsive element in Casperhs (Pirotta, V.(1988). In Vectors: A Survey of Molecular Cloning Vectors and theirUses) was removed to yield Casperhs-1. A 4.3 kb dpp disc specificenhancer (Staehling-Hampton, K., et al.(1994). Cell Growth Differ. 5,585-593) was inserted upstream of the hsp70 basal promoter in Casperhs-1to yield pDMR (dpp mediated reporter). Construct 7 (KUZDN) was alsocloned into pUAST (Brand and Perrimon, 1993) and pCasperhs to generateUAS/KUZDN and hs/KUZDN, respectively. A rough enhancer element(Heberlein et al., 1994) was then inserted into hs/KUZDN to generaterough/KUZDN. Constructs 1 (full length KUZ) and 7 (KUZDN) were alsocloned downstream of the metallothionein promoter in pRMHa-3, a S2 cellexpression vector (Bunch, T. A., et al. (1988) Nucl. Acids Res. 16,1043-1061). The nucleotide coordinates of constructs 1 through 9 are asfollows, using the same numbering as in GenBank accession no. U60591. 1and 8: 723-5630; 2: 723-3578; 3: 723-3462; 4: 723-2757; 5: 1957-2757; 6:1957-5630; 7 and 9: 2757-5630. Note that for all the N- terminaldeletion constructs, a DNA fragment (nucleotides 723-940) containing thesignal peptide was provided at the 5′ end. Site directed mutagenesis wascarried out using Stratagene's QuickChange system.

MKUZDN was generated by an N- terminal truncation that removes the proand catalytic domains of MKUZ. The rest of MKUZ (nucleotide 1483-2573)was ligated either to a DNA fragment (723-940, according to nucleotidecoordinates in U60591) containing the signal peptide of Drosophila KUZto generate MKUZDN-1 or to a fragment (nucleotide 1 -248) containing thesignal peptide of MKUZ to generate MKUZDN-2. MKUZDN-1 was subcloned intopDMR and pUAST for overexpression in Drosophila, and MKUZDN-2 wassubcloned into a modified CS2+ vector (Turner, D. L. and Weintraub, H.(1994). Genes Dev. 8, 1434-1447.) for RNA injection in Xenopus embryos(see below).

Characterization of kuz Homologs from Mouse and Xenopus: PCR primerscorresponding to sequences of a rat gene similar to kuz (GenBankaccession: Z48444) were used to amplify a fragment from a mouse braincDNA library. PCR product was then used to screen oligo(dT) and randomprimed cDNA libraries from the mouse PCC4 cell line (Stratagene). Twooverlapping cDNA, mkuz2 and mkuz3 were characterized and sequenced,which together comprised the whole coding region. mkuz 2 extends fromnucleotide 430 to 2573 and mkuz3 extends from 1 to 1345.

Xenopus kuz was cloned by PCR using degenerate primers (XK1) and (XK4)which correspond to Drosophila KUZ sequence HNFGSPHD (SEQ ID NO:2,residues 609-616) and GYCDVF (SEQ ID NO:2, residues 870-875),respectively. First strand cDNA from stage 18 Xenopus embryos was usedas template in a standard PCR reaction with an annealing temperature of50° C. A PCR product of expected size was purified and used as templatefor another PCR reaction using a nested primer (XK3), corresponding toDrosophila KUZ sequence EECDCG (SEQ ID NO:2, residues 688-693), and XK4.The PCR product was subcloned into Bluescript and sequenced. Anti-senseRNA was used as a probe for whole mount in situ hybridization of Xenopusembryos according to standard procedures (Harland, R. (1991). Meth. CellBiol. 36, 685-695).

For RNA injections in Xenopus embryos, MKUZDN-2 was synthesized in vitrousing SP6 RNA polymerase from a CS2+ vector. Nuclear lacZ RNA wassynthesized from plasmid pSP6nucβGal. 500 pg of MKUZDN RNA, togetherwith 100 pg of lacZ RNA was injected into one blastomere of Xenopusembryos at 2-4 cell stage. lacZ RNA was also injected alone as acontrol. Embryos were fixed at the neural plate stage and stained withRed-Gal (Research Organics, Inc.). Embryos were then processed for insitu hybridization with a neural specific β-tubulin probe.

Drosophila Genetics: For epistasis between kuz and Notch, an activated Nconstruct containing only the cytoplasmic domain of NOTCH (N^(act))under the control of the heatshock promoter (ITM3A insertion on the Xchromosome, from Lieber, T., et al. (1993). Genes Dev. 7, 1949-1965) anda null kuz allele e29-4 (Rooke et al., 1996) were used. Flies of thegenotype ITM3A/+; e29-4 ck FRT40A/+ were crossed to hsFlp/Y; FRT40A. Theprogeny from such a cross were subjected to a one hr heatshock at 38° C.24 to 48 hrs after egg laying to induce kuz mutant clones and anotherone hr heatshock at 7-9 hrs APF to induce the expression of N^(act).Adult flies were processed for scanning electron microscopy and theclones identified by the cell autonomous ck epidermal hair marker as inRooke et al. (1996).

kuz germline clones were generated as in Rooke et al. (1996). Femalesbearing germline clones were mated to e29-4/CyO males. kuz null embryoslacking both maternal and zygotic contribution can be distinguished fromkuz maternal null embryos rescued with one zygotic copy of kuz at lateembryonic stages since kuz null embryos fail to develop any cuticlewhile paternally rescued embryos develop some cuticle structures. kuznull embryos were hand-picked for making protein extracts.

Protein Extracts and Immunoblotting: About 2×10⁶ S2 cells, 50 embryos,or imaginal discs from 16 third instar larvae were used for eachextraction. These materials were homogenized and incubated for 20 min onice in 90 μl of buffer containing 10 mM KC1, 20 mM Tris pH 7.5, 0.1%mercaptoethanol, 1 mM EDTA plus protease and phosphatase inhibitors(leupeptin, aprotinin, PMSF and sodium vanadate). Supernatant wascollected after a low speed spin of 2000 rpm for 5 min. 12 μl ofsupernatant was run on a 6% SDS polyacrylamide gel. Blotting, antibodyincubation, and chemiluminescent detection using the ECL kit were asdescribed in Fehon et al. (1990).

What is claimed:
 1. A method for modulating the interaction of a KUZpolypeptide with a natural KUZ binding target comprising the step ofexposing a mixture of said polypeptide and said binding target to anagent that modulates the binding of said polypeptide to said bindingtarget, said polypeptide comprising an amino acid sequence selected fromthe group consisting of residues 320-673 of SEQ ID NO:2, residues212-454 of SEQ ID NO:4, SEQ ID NO:6, and residues 213-455 of SEQ IDNO:8, wherein the agent is selected from the group consisting of anantibody specific to the polypeptide, a dominant negative fragment ofsaid polypeptide and a metalloprotease inhibitor, and the binding targetis a protease substrate specifically cleaved by the polypeptide.
 2. Amethod according to claim 1, wherein the amino acid sequence is residues320-673 of SEQ ID NO:2.
 3. A method according to claim 1, wherein theamino acid sequence is residues 212-454 of SEQ ID NO:4.
 4. A methodaccording to claim 1, wherein the amino acid sequence is SEQ ID NO:6. 5.A method according to claim 1, wherein the amino acid sequence isresidues 213-455 of SEQ ID NO:8.
 6. A method according to claim 1,wherein the amino acid sequence is SEQ ID NO:2.
 7. A method according toclaim 1, wherein the amino acid sequence is SEQ ID NO:4.
 8. A methodaccording to claim 1, wherein the amino acid sequence is SEQ ID NO:8.